Abstract The objective of this proposal is to illuminate epigenetic transcriptional regulation during mouse embryogenesis and stem cells through investigations of the Polycomb group. The Polycomb group comprises a prominent set of histone modifiers that are essential for the execution of diverse developmental processes, including X- chromosome inactivation, self-renewal and differentiation of embryonic stem cells, cell and tissue specification, and body patterning in mammals. The Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27me3 through the methyltransferases EZH2 and its paralog EZH1. H3K27me3 functions as a key epigenetic mark in development and is dysregulated in human diseases. Through much work in Drosophila, PRC2-catalyzed H3K27me3 has been shown to epigenetically maintain transcriptional silencing. Through preliminary investigations in early mouse embryos, we propose that PRC2 may also initiate transcriptional silencing. In Aim 1, we will therefore test the role of PRC2 in initiating epigenetic transcriptional silencing. We have further found the PRC2 protein EED can function independently of PRC complexes to execute epigenetic silencing in early embryos and in embryo embryo-derived stem cells. In Aim 2, we will define the non-PRC role of EED in epigenetic silencing in preimplantation mouse embryos and its derived stem cells. Finally, countering the prevailing dogma that H3K27me3 is deposited solely by PRC2 our results demonstrate an additional H3K27me3 catalyst. In Aim 3, we propose to identify and dissect the function of a novel H3K27me3 catalyst in extra-embryonic and embryonic stem cells. All three Aims utilize unbiased approaches to define novel functions and mechanisms of PRC2 proteins and H3K27me3 catalysis during mouse embryogenesis and in early embryo-derived stem cells. Our central hypothesis is that the mode of epigenetic regulation ascribed to the Polycomb group can occur via alternate mechanisms and proteins. The expected findings will increase our understanding of the epigenetic logic underlying embryonic development and how epigenetic dysregulation contributes to human disease.